The analysis of aqueous liquids to determine the presence or concentration of one or more components of the liquid, hereinafter designated analyte(s), is extremely important. Such analyses are carried out in a wide variety of situations including, among others, analyses of aqueous industrial liquids and analyses of aqueous liquids of biological origin such as are performed in hundreds of doctors' offices, clinics, and hospitals each day. Increasing numbers of these analyses are now being performed in whole or in part, by use of enzymatic reagents. For example, the use of glucose oxidase in serum glucose tests has become widely accepted as a standard method for determination of serum glucose.
Many of the enzymatic reagents used in these various liquid analysis procedures are derived from microbiological organisms. Although many, if not most, of these enzymatic reagents are highly specific in terms of their "catalytic activity", these enzymes are typically complex mixtures of biochemical substances. Therefore, these enzymatic reagents are difficult to characterize on a molecular chemical basis and are also difficult to completely purify and isolate. Not infrequently, many of these enzymatic reagents contain undesired amounts of other enzymes, the detection and removal of which involves costly purification and isolation procedures. Accordingly, in many cases, addition of an "inhibitor" to eliminate or substantially reduce unwanted enzymatic activity in a particular liquid analysis procedure employing an enzymatic reagent can be much less costly and more convenient. The inhibitor functions to physically or chemically block or at least substantially reduce the enzymatic activity of the undesired enzyme.
U.S. Pat. No. 3,956,069 issued May 11, 1976 discloses enzymatic assays for determining the presence or concentration of several different serum analytes including glucose and creatine phosphokinase by use of nicotinamide adenine dinucleotide (NAD) or the reduced form of nicotinamide adenine dinucleotide (NADH). The patent discloses that the enzyme lactate dehydrogenase (LDH) interferes with these enzymatic assays because of the following reaction which is catalyzed by LDH: ##STR1##
As shown in the above-identified reaction, if there is any lactate or lactic acid (lactic acid being converted to its lactate form at any pH greater than about 5), the presence of the enzyme LDH may cause the lactate to interfere with the assay. That is, LDH, due to its activity on lactate, can also catalyze conversion of NAD to NADH and thereby contribute a source of error to an enzymatic assay which is based on the NAD-NADH reaction couple. To eliminate the source of this interference, U.S. Pat. No. 3,956,069 discloses that oxalic acid, oxamic acid, or their salts, can be added to the enzymatic reagent to inhibit the action of the LDH enzyme on lactate.
U.S. Pat. No. 3,956,069 describes no method for the inhibition of the enzyme lactate oxidase. The presence of lactate oxidase in an enzymatic reagent can provide a major interferent in many liquid analysis procedures in which lactate or lactic acid is also present. This is because many liquid analysis procedures employ an enzymatic reaction sequence in which hydrogen peroxide is produced and because lactate oxidase catalyzes the following reaction: ##STR2##
Thus, enzymatic, "hydrogen peroxide-linked" liquid analyses, i.e., those anlayses that determine the presence or concentration of a selected analyte by an enzymatic reaction sequence in which hydrogen peroxide is produced, can clearly be interferred with by the action of any lactate oxidase present in the enzymatic reagent whenever these analyses are performed on aqueous liquids containing, in addition to the selected analyte, lactate or lactic acid. Lactate or lactic acid is generally present in serum and whole blood, and therefore will produce interference in enzymatic hydrogen peroxide-linked assays performed on serum or whole blood samples using enzymatic reagent compositions containing lactate oxidase in addition to the desired enzymatic reagents. Accordingly, the discovery of an inhibitor for lactate oxidase would represent a highly useful contribution to the art.